Blood contains several cell populations. The majority of them are erythrocytes and platelets. Erythrocytes function in the exchange and transport of carbon dioxide and oxygen. The platelets play a role in blood coagulation. The leukocytes are a minor population involved in the control of the immune system.
By microscopic analysis or by flow cytometric analysis, one can distinguish three subpopulations in the leukocyte population: polynuclear cells, having several nuclei and mononuclear cells having one nucleus, among which one can further distinguish lymphocytes with a small, round nucleus and monocytes with a larger moon-shaped nucleus.
Using cytometry, in a scatter analysis, one can distinguish these populations in a scattergram. In the leukocyte subpopulation, approximately 60% of the cells are polynuclear cells, approximately 30% are lymphocytes and approximately 10% are monocytes. Variation of the percentages of these subpopulations can be an indication of the state of health of an individual.
By the staining of blood cells with monoclonal antibodies conjugated to a fluorescent marker and after analysis of cells by microscopy or by flow cytometry one can distinguish blood cells and subpopulations more precisely and in more detail than by simple optic means.
For instance, using the marker CD19, one can distinguish B-cells in the lymphocyte population which are derived from bone marrow. In the same population one can distinguish, with the CD3 marker, T-cells which are derived from the thymus. With the CD4 and CD8 markers one can distinguish T-cells with a helper function or a suppressor/killer function, respectively.
The identification of lymphocyte sub-populations is important for the diagnosis or the treatment of diseases of the immune system.
Because the erythrocytes are a major population in the blood, they can mask leukocytes and make their analysis by flow cytometry difficult. Conventional immunofluorescence techniques include a physical separation of lymphocytes and erythrocytes, for instance by gradient density centrifugation (Boyem, A. 1968 Scand. J. Clin. Lab. Invest., 21 suppl. 97).
Another method, more rapid, is erythrocyte lysis in whole blood. For instance, in the method of Hansen (U.S. Pat. No. 4.284.412, EP-A-0.022.670), a sample of blood treated with an anticoagulant is mixed with a fluorescent antibody conjugate preparation. After incubation and lysis of the erythrocytes, the sample is passed in a flow cytometer, to analyze leukocyte populations which are positive for a certain antibody.
To perform a correct cytometric analysis, it is not only necessary to lyse all the erythrocytes, and one also has to conserve all the leukocytes in a morphological state in which the cytometer is capable of distinguishing between polynuclear cells, monocytes, lymphocytes and the mixture of cellular debris and platelets. Various methods of lysing erythrocytes exist. These methods may be based, for instance, on acid treatment, on alkaline treatment, treatment based on the use of ammonium chloride, polyhydric alcohols or on hypotonic shock. A problem with all these methods is that in lysing the erythrocytes, modifications are introduced in the leukocyte morphology.
In conventional methods, one limits the leukocyte degradation, for instance by analyzing the sample immediately after the lysis or by neutralization after acid and hypotonic conditions, or by washing the cells, or by addition of a fixing reagent such as formaldehyde or paraformaldehyde.
The methods of lysis used by the manufacturers of cytometers are adapted to the optical characteristics of the different cytometers. The cytometers of Becton, Dickinson and Co. are relatively insensitive to morphological changes of leukocytes. A lysis under slightly hypotonic conditions is indicated for this type of apparatus (U.S. Pat. No. 4.902.613). Cytometers of the Coulter company are much more sensitive to morphologic modification of the leukocytes. A lysing system from Coulter (WO-89/0509) applies hypotonic and acid lysis during six seconds approximately and corrects thereafter the isotonic value and the pH of the mixture. The inconvenience of such a system is that the exact six-second period is difficult to achieve under non-automatic conditions of operation.
Another method consists of the use of ammonium chloride. In this method, the ionic strength of the solution is physiologic and the leukocyte morphology is well conserved after a short period. The disadvantage of this method is that the ammonium chloride causes an intracellular rise of pH and a continuous degradation of leukocytes, even after the washing of the cells, which cannot be arrested with a fixing reagent.
It would be desirable to have a method of leukocyte analysis and of erythrocyte lysis which avoids washing of the samples and, therefore, inaccurate leukocyte counts.